The 135 kb amplified dihydrofolate reductase (DHFR) replicon from methotrexate-resistant Chinese hamster ovary CHOC 400 cells has been cloned, and a large portion of the replicon has been mapped. Pulse-labeling experiments in synchronized cells have shown that initiation of replication within each amplified replicon commences at a specific site approximately 25 kb downstream from the 3' end of the DHFR gene. The genomic DNA representing the initiation locus was cloned as a 28 kb insert in the cosmid vector, pHC79. This proposal is directed toward precisely locating the nucleotide sequence that functions as the origin of replication within this region. To accomplish this, cytosine arabinoside will be used to limit DNA synthesis at the onset of S to regions of the CHOC 400 genome that include and surround origins of replication. Nascent DNA from initiation sites will be isolated from replication-arrested CHOC 400 cells, labeled at the 5' end with 32P, and hybridized to single-stranded DNA from rucombinant M13 clones derived from the DHFR initiation region. The labeled nascent DNA-M13 hybrids will then be cut with restriction enzymes prior to analysis of the cleavage products on sequencing gels. This technique will unambiguously identify the nucleotides at which polymerization of nascent DNA in whole cells is initiated. The primary necleotide sequence of this functionally-defined origin of replication will be determined, and the sequence will be examined by computer analysis for regulatory elements and secondary structures. The distribution of this sequence throughout the Chinese hamster genome will be determined, and experiments will be conducted to determine if all regions containing the sequence are co-ordinately activated at the onset of S. Clones of the original sequence will also be used as reagents for the identification of factors, particularly proteins, that interact with origins at the onset of S. Isolation and characterization of the sequences utilized in whole cells as chromosomal origins of replication should permit detailed examination of the control of initiation of DNA synthesis at the molecular level.